In the intervertebral disc, the cells of the nucleus pulposus (NP) function in a unique environmental niche, characterized by a very limited vascular supply that imposes metabolic constraints on the disc cells and a proteoglycan-rich extracellular matrix. We have shown that the oxygen tension of the NP is very low and unlike all other tissues, cells of the NP exhibit constitutive expression of HIF-1a. We advance the new hypothesis that constitutive expression of HIF-1a serves to adapt the NP cells to their avascular environment and that changes in HIF-1 activity is required for their survival and function. The first Specific Aims is to test the hypotheses that high constitutive HIF-1a expression is required for survival of the NP cells in the hypoxic disc; to investigate mechanisms regulating constitutive expression and activity of HIF-1a; using tissue derived from human degenerative discs to correlate NP HIF-1 activity with degeneration and susceptibility to apoptosis. We will transiently and stably suppress HIF-1a and assess the impact of HIF-1a downregulation on cell survival and death. We will then restore HIF-1a expression in suppressed cells and evaluate survival, apoptosis and cell phenotype. We will also determine if changes in expression of pVHL, FIH and PHDs regulate HIF-1a expression and transcriptional activity in NP cells. We will use cells isolated form clinical human disc samples, with varying degree of degeneration, to determine if susceptibility to apoptogen treatment is linked to endogenous HIF-1a activity and to the level of degeneration. The second specific aim is to test the hypothesis that constitutive HIF-1a expression regulates proteoglycan synthesis and GAG chain formation and sulfation; to correlate HIF-1 activity to proteoglycan synthesis in human degenerative disc samples. We will determine the role of constitutive HIF-1a in the expression and synthesis of aggrecan core protein, a molecule that has a HIF-1 responsive promoter element. In addition, we will evaluate the role of HIF-1a in regulating expression of enzymes required for GAG synthesis (glucuronosyltransferase I; GlcAT-I) and chondroitin sulfation (chondroitin-4-O-sulfosyl transferase 2; C4ST-2). We will investigate if proteoglycan synthesis by cells isolated form clinical human disc samples is linked to HIF-1a activity. The last Specific Aim is to develop a rabbit model with suppression of HIF-1a in NP cells using lentiviral HIF-1a-SiRNA; to learn if inactivation of HIF-1a promotes a loss of cellularity, a decrease in proteoglycan synthesis and degeneration of the intervertebral disc. To complement the in vivo studies, and to investigate early cellular events in NP degeneration, we will perform disc organ culture studies using floxed-HIF-1a mouse. Inactivation of the HIF-1a gene in organ cultured discs will be achieved by injecting adenovirus expressing cre recombinase. The information provided by proposed studies will provide insights into mechanisms of early degenerative changes in the NP cells and provide foundation for development of interventional strategies to prevent degenerative disc disease. PUBLIC HEALTH RELEVANCE The incidence of low back pain, which is often linked to degenerative changes in the intervertebral disc, is extraordinary high with annual costs to the US health care industry exceeding 33 billion dollars. None of the current therapies can completely restore the function of the degenerative intervertebral disc and thereby prevent further deterioration of the compromised spine. The proposed studies will provide insights into mechanisms of early degenerative changes in the disc and may permit development of interventional strategies to prevent degenerative disease.